Epidemiologic studies imply that sequential infections with distinct dengue virus (DEN) serotypes are associated with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Understanding the basis for this phenomenon is crucial for developing therapies and a safe dengue vaccine. The lack of a small animal model has greatly hampered the elucidation of the mechanism of severe dengue. Our preliminary data suggest that DEN tropism early in infection may be similar in mice and humans. Thus, we propose to use a murine model to study primary (1[unreadable]) and secondary (2[unreadable]) DEN infection, using cellular tropism and viral load/kinetics as endpoints. Since the cellular tropism of DEN in humans is not well defined, we will apply techniques developed to study murine DEN infection to detect the cellular localization of replicating virus in humans through autopsy studies. The goal of Specific Aim 1 is to identify the cellular targets of DEN infection in mice and evaluate parameters that influence cellular tropism and viral load. We will define the cellular tropism of DEN in mice and determine how cellular tropism, viral load, and kinetics of DEN infection are affected by virus serotype and age and strain of mice during 1[unreadable] DEN infection. This knowledge will provide an alternative endpoint in the mouse model and will be used to assess alterations in 2[unreadable] DEN infections. In Specific Aim 2, we will develop a model for secondary DEN infection and evaluate changes in the in vivo cellular tropism, viral load, kinetics of DEN infection, and immune response. Several approaches will be used, including 1) sequential infections of a single mouse-by different DEN serotypes, 2) passive transfer of DEN-immune sera to naTve mice to mimic a 1[unreadable] infection followed by challenge with another DEN serotype, and 3) transfer of maternal antibodies, followed by DEN infection of offspring. After 2[unreadable] infection, mice will be evaluated according to histopathologic, virologic, immunologic, and clinical parameters. Cellular targets of DEN infection in fatal human DHF/DSS cases will be identified via autopsy studies in Specific Aim 3. In collaboration with the Ecuadorian Ministry of Health, techniques developed in Aim 1 will be applied to determine the cellular localization of DEN antigen and RNA in human autopsies. Immunohistochemistry will be performed on various tissues to identify cell type and presence of replicating DEN. Together, these investigations will provide useful tools and critical data regarding the in vivo cellular tropism of DEN, a category A Priority Pathogen, in both mice and fatal human cases. They will also generate a model system for studying the effects of primary and sequential infections of DEN and potentially other flaviviruses.